Compounds for inhibiting KSP kinesin activity

ABSTRACT

The present invention relates to compounds of Formula I or pharmaceutically acceptable salts or solvates thereof: 
                         
The present invention provides compositions comprising these compounds that are useful for treating cellular proliferative diseases or disorders associated with KSP kinesin activity and for inhibiting KSP kinesin activity.

This Application claims the benefit of U.S. Provisional Application Ser.No. 60/659,973, filed Mar. 9, 2005, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compound and compositions that areuseful for treating cellular proliferative diseases or disordersassociated with Kinesin Spindle Protein (“KSP”) kinesin activity and forinhibiting KSP kinesin activity.

BACKGROUND OF THE INVENTION

Cancer is a leading cause of death in the United States and throughoutthe world. Cancer cells are often characterized by constitutiveproliferative signals, defects in cell cycle checkpoints, as well asdefects in apoptotic pathways. There is a great need for the developmentof new chemotherapeutic drugs that can block cell proliferation andenhance apoptosis of tumor cells.

Conventional therapeutic agents used to treat cancer include taxanes andvinca alkaloids, which target microtubules. Microtubules are an integralstructural element of the mitotic spindle, which is responsible for thedistribution of the duplicated sister chromatids to each of the daughtercells that result from cell division. Disruption of microtubules orinterference with microtubule dynamics can inhibit cell division andinduce apoptosis.

However, microtubules are also important structural elements innon-proliferative cells. For example, they are required for organelleand vesicle transport within the cell or along axons. Sincemicrotubule-targeted drugs do not discriminate between these differentstructures, they can have undesirable side effects that limit usefulnessand dosage. There is a need for chemotherapeutic agents with improvedspecificity to avoid side effects and improve efficacy.

Microtubules rely on two classes of motor proteins, the kinesins anddyneins, for their function. Kinesins are motor proteins that generatemotion along microtubules. They are characterized by a conserved motordomain, which is approximately 320 amino acids in length. The motordomain binds and hydrolyses ATP as an energy source to drive directionalmovement of cellular cargo along microtubules and also contains themicrotubule binding interface (Mandelkow and Mandelkow, Trends CellBiol. 2002, 12:585-591).

Kinesins exhibit a high degree of functional diversity, and severalkinesins are specifically required during mitosis and cell division.Different mitotic kinesins are involved in all aspects of mitosis,including the formation of a bipolar spindle, spindle dynamics, andchromosome movement. Thus, interference with the function of mitotickinesins can disrupt normal mitosis and block cell division.Specifically, the mitotic kinesin KSP (also termed EG5), which isrequired for centrosome separation, was shown to have an essentialfunction during mitosis. Cells in which KSP function is inhibited arrestin mitosis with unseparated centrosomes (Blangy et al., Cell 1995,83:1159-1169). This leads to the formation of a monoastral array ofmicrotubules, at the end of which the duplicated chromatids are attachedin a rosette-like configuration. Further, this mitotic arrest leads togrowth inhibition of tumor cells (Kaiser et al., J. Biol. Chem. 1999,274:18925-18931). Inhibitors of KSP would be desirable for the treatmentof proliferative diseases, such as cancer.

Kinesin inhibitors are known, and several molecules have recently beendescribed in the literature. For example, adociasulfate-2 inhibits themicrotubule-stimulated ATPase activity of several kinesins, includingCENP-E (Sakowicz et al., Science 1998, 280:292-295). Rose Bengallactone, another non-selective inhibitor, interferes with kinesinfunction by blocking the microtubule binding site (Hopkins et al,Biochemistry (2000) 39:2805-2814). Monastrol, a compound that has beenisolated using a phenotypic screen, is a selective inhibitor of the KSPmotor domain (Mayer et al., Science 1999, 286:971-974). Treatment ofcells with monastrol arrests cells in mitosis with monopolar spindles.

KSP, as well as other mitotic kinesins, are attractive targets for thediscovery of novel chemotherapeutics with anti-proliferative activity.There is a need for compounds useful in the inhibition of KSP, and inthe treatment of proliferative diseases, such as cancer.

SUMMARY OF THE INVENTION

The present invention relates to a compound represented by Formula I:

or a salt or solvate thereof.

In one embodiment, the present invention provides a pharmaceuticalformulation or a composition comprising the above compound or a salt orsolvate thereof and a pharmaceutically acceptable carrier, adjuvant orvehicle. In another embodiment, methods for the treatment of cellularproliferative diseases, disorders associated with KSP kinesin activityand/or for inhibiting KSP kinesin activity in a subject comprisingadministering a therapeutically effective amount of the above compoundand a pharmaceutically acceptable carrier, adjuvant or vehicle to thesubject are also provided.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients, reaction conditions, andso forth used in the specification and claims are to be understood asbeing modified in all instances by the term “about.”

DETAILED DESCRIPTION

In one embodiment, the present invention relates to relates to acompound represented by Formula I:

or a pharmaceutically acceptable salt or solvate thereof.

In other embodiments, the present invention provides processes forproducing the inventive compound, pharmaceutical formulations orcompositions comprising the compound, and methods of treating orpreventing one or more conditions or diseases associated with KSPkinesin activity such as those discussed in detail below.

As used above, and throughout the specification, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings:

“Subject” includes both mammals and non-mammalian animals.

“Mammal” includes humans and other mammalian animals.

The term “substituted” means that one or more hydrogens on thedesignated atom is replaced with a selection from the indicated group,provided that the designated atom's normal valency under the existingcircumstances is not exceeded, and that the substitution results in astable compound. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds. By“stable compound” or “stable structure” is meant a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

The term “optionally substituted” means optional substitution with thespecified groups, radicals or moieties. It should be noted that any atomwith unsatisfied valences in the text, schemes, examples and tablesherein is assumed to have the hydrogen atom(s) to satisfy the valences.

Also included in the scope of this invention are oxidized forms of theheteroatoms (e.g., nitrogen and sulfur) that are present in the compoundof this invention. Such oxidized forms include N(O) [N⁺—O⁻], S(O) andS(O)₂.

When a functional group in a compound is termed “protected”, this meansthat the group is in modified form to preclude undesired side reactionsat the protected site when the compound is subjected to a reaction.Suitable protecting groups will be recognized by those with ordinaryskill in the art as well as by reference to standard textbooks such as,for example, T. W. Greene et al, Protective Groups in organic Synthesis(1991), Wiley, New York.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

Isomers of the compound of Formula I, including enantiomers,stereoisomers, rotamers, tautomers, diastereomers, and racemates arealso contemplated as being part of this invention. The inventionincludes d and I isomers in both pure form and in admixture, includingracemic mixtures. Isomers can be prepared using conventional techniques,either by reacting optically pure or optically enriched startingmaterials or by separating isomers of the compound of the Formula I.Isomers may also include geometric isomers, e.g., when a double bond ispresent. Polymorphous forms of the compound of Formula I, whethercrystalline or amorphous, also are contemplated as being part of thisinvention. The (+) isomer of the present compound are preferred.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C— or ¹⁴C-enriched carbonare also within the scope of this invention.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in alternative tautomeric forms. All suchtautomeric forms of the present compounds are within the scope of theinvention. Unless otherwise indicated, the representation of eithertautomer is meant to include the other. For example, both isomers (1)and (2) are contemplated:

wherein R′ is H or C₁₋₆ unsubstituted alkyl.

Prodrugs and solvates of the compound of the invention are alsocontemplated herein. The term “prodrug”, as employed herein, denotes acompound that is a drug precursor which, upon administration to asubject, undergoes chemical conversion by metabolic or chemicalprocesses to yield the compound of formula I or a salt or solvatethereof (e.g., a prodrug on being brought to the physiological pH orthrough enzyme action is converted to the desired drug form). Adiscussion of prodrugs is provided in T. Higuchi and V. Stella,Pro-drugs as Novel Delivery Systems (1987) Volume 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in Drug Design, (1987)Edward B. Roche, ed., American Pharmaceutical Association and PergamonPress, both of which are incorporated herein by reference thereto.

“Solvate” means a physical association of a compound of this inventionwith one or more solvent molecules. This physical association involvesvarying degrees of ionic and covalent bonding, including hydrogenbonding. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates, and the like.“Hydrate” is a solvate wherein the solvent molecule is H₂O.

The compound of the invention may also exist as, or optionally convertedto, a solvate. Preparation of solvates is generally known. Thus, forexample, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601-611 (2004)describe the preparation of the solvates of the antifungal fluconazolein ethyl acetate as well as from water. Similar preparations ofsolvates, hemisolvate, hydrates and the like are described by E. C. vanTonder et al, AAPS PharmSciTech., 5(1), article 12 (2004); and A. L.Bingham et al, Chem. Commun., 603-604 (2001). A typical, non-limiting,process involves dissolving a compound in desired amounts of the desiredsolvent (organic or water or mixtures thereof) at a higher than ambienttemperature, and cooling the solution at a rate sufficient to formcrystals which are then isolated by standard methods. Analyticaltechniques such as, for example I. R. spectroscopy, show the presence ofthe solvent (or water) in the crystals as a solvate (or hydrate).

“Effective amount” or “therapeutically effective amount” is meant todescribe an amount of a compound or a composition of the presentinvention effective in inhibiting mitotic kinesins, in particular KSPkinesin activity, and thus producing the desired therapeutic,ameliorative, inhibitory or preventative effect in a suitable subject.

The compound of formula I can form salts which are also within the scopeof this invention. Reference to the compound of formula I herein isunderstood to include reference to salts, and solvates thereof, unlessotherwise indicated. The term “salt(s)”, as employed herein, denotesacidic salts formed with inorganic and/or organic acids, as well asbasic salts formed with inorganic and/or organic bases. In addition,when the compound of formula I contains both a basic moiety, such as,but not limited to a pyridine or imidazole, and an acidic moiety, suchas, but not limited to a carboxylic acid, zwitterions (“inner salts”)may be formed and are included within the term “salt(s)” as used herein.Pharmaceutically acceptable (i.e., non-toxic, physiologicallyacceptable) salts are preferred, although other salts are also useful.Salts of the compound of the formula I may be formed, for example, byreacting the compound of formula I with an amount of acid or base, suchas an equivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization. Acids(and bases) which are generally considered suitable for the formation ofpharmaceutically useful salts from basic (or acidic) pharmaceuticalcompounds are discussed, for example, by S. Berge et al, Journal ofPharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. ofPharmaceutics (1986) 33 201-217; Anderson et al, The Practice ofMedicinal Chemistry (1996), Academic Press, New York; in The Orange Book(Food & Drug Administration, Washington, D.C. on their website); and P.Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of PharmaceuticalSalts: Properties, Selection, and Use, (2002) Int'l. Union of Pure andApplied Chemistry, pp. 330-331. These disclosures are incorporatedherein by reference thereto.

Exemplary acid addition salts include acetates, adipates, alginates,ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates,borates, butyrates, citrates, camphorates, camphorsulfonates,cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides,hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates,methanesulfonates, methyl sulfates, 2-naphthalenesulfonates,nicotinates, nitrates, oxalates, pamoates, pectinates, persulfates,3-phenylpropionates, phosphates, picrates, pivalates, propionates,salicylates, succinates, sulfates, sulfonates (such as those mentionedherein), tartarates, thiocyanates, toluenesulfonates (also known astosylates,) undecanoates, and the like.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, aluminum salts, zinc salts, salts withorganic bases (for example, organic amines) such as benzathines,diethylamine, dicyclohexylamines, hydrabamines (formed withN,N-bis(dehydroabietyl) ethylenediamine), N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, piperazine,phenylcyclohexylamine, choline, tromethamine, and salts with amino acidssuch as arginine, lysine and the like. Basic nitrogen-containing groupsmay be quarternized with agents such as lower alkyl halides (e.g.methyl, ethyl, propyl, and butyl chlorides, bromides and iodides),dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates),long chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides), aralkyl halides (e.g. benzyl and phenethylbromides), and others.

All such acid salts and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the invention. All acid and basesalts, as well as solvates, are considered equivalent to the free formsof the corresponding compounds for purposes of the invention.

Generally, the compound of Formula I is either commercially available orcan be prepared by a variety of methods well known to those skilled inthe art, for example, by the methods as outlined in Scheme 1 below:

wherein R¹ is t-butyl.

The compound of the invention can be useful in a variety of applicationsinvolving alteration of mitosis. As will be appreciated by those skilledin the art, mitosis may be altered in a variety of ways; that is, onecan affect mitosis either by increasing or decreasing the activity of acomponent in the mitotic pathway. Mitosis may be affected (e.g.,disrupted) by disturbing equilibrium, either by inhibiting or activatingcertain components. Similar approaches may be used to alter meiosis.

In a particular embodiment, the compound of the invention can be used toinhibit mitotic spindle formation, thus causing prolonged cell cyclearrest in mitosis. By “inhibit” in this context is meant decreasing orinterfering with mitotic spindle formation or causing mitotic spindledysfunction. By “mitotic spindle formation” herein is meant organizationof microtubules into bipolar structures by mitotic kinesins. By “mitoticspindle dysfunction” herein is meant mitotic arrest and monopolarspindle formation.

The compound of the invention is useful for binding to, and/orinhibiting the activity of, a mitotic kinesin, KSP. In one embodiment,the KSP is human KSP, although the compounds may be used to bind to orinhibit the activity of KSP kinesins from other organisms. In thiscontext, “inhibit” means either increasing or decreasing spindle poleseparation, causing malformation, i.e., splaying, of mitotic spindlepoles, or otherwise causing morphological perturbation of the mitoticspindle. Also included within the definition of KSP for these purposesare variants and/or fragments of KSP (see U.S. Pat. No. 6,437,115). Inaddition, the present inventive compound is useful for binding to ormodulating other mitotic kinesins.

The compound of the invention can be used to treat cellularproliferation diseases. Such disease states which can be treated by thecompound, compositions and methods provided herein include, but are notlimited to, cancer (further discussed below), hyperplasia, cardiachypertrophy, autoimmune diseases, fungal disorders, arthritis, graftrejection, inflammatory bowel disease, immune disorders, inflammation,cellular proliferation induced after medical procedures, including, butnot limited to, surgery, angioplasty, and the like. Treatment includesinhibiting cellular proliferation. It is appreciated that in some casesthe cells may not be in a hyper- or hypoproliferation state (abnormalstate) and still require treatment. For example, during wound healing,the cells may be proliferating “normally”, but proliferation enhancementmay be desired. Thus, in one embodiment, the invention herein includesapplication to cells or subjects afflicted or subject to impendingaffliction with any one of these disorders or states.

The compound, compositions and methods provided herein can beparticularly useful for the treatment of cancer including solid tumorssuch as skin, breast, brain, colon, gall bladder, thyroid, cervicalcarcinomas, testicular carcinomas, etc. More particularly, cancers thatmay be treated by the compounds, compositions and methods of theinvention include, but are not limited to:

Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma;

Lung: bronchogenic carcinoma (squamous cell, undifferentiated smallcell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma;

Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma);

Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor(nephroblastoma), lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);

Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;

Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors;

Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma (pinealoma), glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma);

Gynecological: uterus (endometrial carcinoma), cervix (cervicalcarcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma(serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma);

Hematologic: blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, acute and chronic lymphocytic leukemia,myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignantlymphoma), B-cell lymphoma, T-cell lymphoma, hairy cell lymphoma,Burkeft's lymphoma, promyelocytic leukemia;

Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma,Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis;

Adrenal glands: neuroblastoma; and

Other tumors: including xenoderoma pigmentosum, keratoctanthoma andthyroid follicular cancer.

As used herein, treatment of cancer includes treatment of cancerouscells, including cells afflicted by any one of the above-identifiedconditions.

The compound of the present invention may also be useful in thechemoprevention of cancer. Chemoprevention is defined as inhibiting thedevelopment of invasive cancer by either blocking the initiatingmutagenic event or by blocking the progression of pre-malignant cellsthat have already suffered an insult or inhibiting tumor relapse.

The compound of the present invention may also be useful in inhibitingtumor angiogenesis and metastasis.

The compound of the present invention may also be useful as antifungalagents, by modulating the activity of the fungal members of the bimCkinesin subgroup, as is described in U.S. Pat. No. 6,284,480.

The present compound can also be used in combination with other knowntherapeutic agents and anti-cancer agents. Combinations of the presentinventive compound with one or more other anti-cancer orchemotherapeutic agents are within the scope of the invention. Examplesof such other anti-cancer or chemotherapeutic agents can be found inCancer Principles and Practice of Oncology by V. T. Devita and S.Hellman (editors), 6^(th) edition (Feb. 15, 2001), Lippincott Williams &Wilkins Publishers. A person of ordinary skill in the art would be ableto discern which combinations of agents would be useful based on theparticular characteristics of the drugs and the cancer involved. Suchanti-cancer agents include, but are not limited to, the following:estrogen receptor modulators, androgen receptor modulators, retinoidreceptor modulators, cytotoxic/cytostatic agents, antiproliferativeagents, prenyl-protein transferase inhibitors, HMG-CoA reductaseinhibitors and other angiogenesis inhibitors, inhibitors of cellproliferation and survival signaling, apoptosis inducing agents andagents that interfere with cell cycle checkpoints. The present compoundcan also be useful when co-administered with radiation therapy.

The phrase “estrogen receptor modulators” refers to compounds thatinterfere with or inhibit the binding of estrogen to the receptor,regardless of mechanism. Examples of estrogen receptor modulatorsinclude, but are not limited to, tamoxifen, raloxifene, idoxifene,LY353381, LY117081, toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-ydrazone, aid SH646.

The phrase “androgen receptor modulators” refers to compounds whichinterfere or inhibit the binding of androgens to the receptor,regardless of mechanism. Examples of androgen receptor modulatorsinclude finasteride and other 5α-reductase inhibitors, nilutamide,flutamide, bicalutamide, liarozole, and abiraterone acetate.

The phrase “retinoid receptor modulators” refers to compounds whichinterfere or inhibit the binding of retinoids to the receptor,regardless of mechanism. Examples of such retinoid receptor modulatorsinclude bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, a difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

The phrase “cytotoxic/cytostatic agents” refer to compounds which causecell death or inhibit cell proliferation primarily by interferingdirectly with the cell's functioning or inhibit or interfere with cellmycosis, including alkylating agents, tumor necrosis factors,intercalators, hypoxia activatable compounds, microtubuleinhibitors/microtubule-stabilizing agents, inhibitors of mitotickinesins, inhibitors of kinases involved in mitotic progression,antimetabolites; biological response modifiers; hormonal/anti-hormonaltherapeutic agents, haematopoietic growth factors, monoclonal antibodytargeted therapeutic agents, monoclonal antibody therapeutics,topoisomerase inhibitors, proteasome inhibitors and ubiquitin ligaseinhibitors.

Examples of cytotoxic agents include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide (TEMODAR™ from Schering-Plough Corporation,Kenilworth, N.J.), cyclophosphamide, heptaplatin, estramustine,improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride,pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, doxorubicin,irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride,diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deansino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755,4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunombicin (see WO00/50032), methoxtrexate, gemcitabine.

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteasome inhibitors include, but are not limited to,lactacystin and bortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxel, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine,(5a,5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2-(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,dimesna, and camptostar.

Other useful anti-cancer agents that can be used in combination with thepresent compound include thymidilate synthase inhibitors, such as5-fluorouracil.

In one embodiment, inhibitors of mitotic kinesins include, but are notlimited to, inhibitors of KSP, inhibitors of MKLP1, inhibitors ofCENP-E, inhibitors of MCAK, inhibitors of Kif14, inhibitors of Mphosph1and inhibitors of Rab6-KIFL.

The phrase “inhibitors of kinases involved in mitotic progression”include, but are not limited to, inhibitors of aurora kinase, inhibitorsof Polo-like kinases (PLK) (in particular inhibitors of PLK-1),inhibitors of bub-1 and inhibitors of bub-R1.

The phrase “antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar®.

Examples of monoclonal antibody therapeutics useful for treating cancerinclude Erbitux® (Cetuximab).

The phrase “HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductaseinhibitors that may be used include but are not limited to lovastatin(MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039),simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®;see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164,5,118,853, 5,290,946 and 5,356,896) and atorvastatin (LIPITOR®; see U.S.Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The structuralformulas of these and additional HMG-CoA reductase inhibitors that maybe used in the instant methods are described at page 87 of M. Yalpani,“Cholesterol Lowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb.1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoAreductase inhibitor as used herein includes all pharmaceuticallyacceptable lactone and open-acid forms (i.e., where the lactone ring isopened to form the free acid) as well as salt and ester forms ofcompounds which have HMG-CoA reductase inhibitory activity, andtherefore the use of such salts, esters, open acid and lactone forms isincluded in the scope of this invention.

The phrase “prenyl-protein transferase inhibitor” refers to a compoundwhich inhibits any one or any combination of the prenyl-proteintransferase enzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-Il (GGPTase-II, also called RabGGPTase).

Examples of prenyl-protein transferase inhibitors can be found in thefollowing publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510,5,589,485, 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604181, European Patent Publ. 0696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO, 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European ofCancer, Vol. 35, No. 9, pp. 1394-1401(1999).

Examples of farnesyl protein transferase inhibitors includeSARASAR™(4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoethyl]-1-piperidinecarboxamidefrom Schering-Plough Corporation, Kenilworth, N.J.), tipifarnib(Zarnestra® or R115777 from Janssen Pharmaceuticals), L778,123 (afarnesyl protein transferase inhibitor from Merck & Company, WhitehouseStation, N.J.), BMS 214662 (a farnesyl protein transferase inhibitorfrom Bristol-Myers Squibb Pharmaceuticals, Princeton, N.J.).

The phrase “angiogenesis inhibitors” refers to compounds that inhibitthe formation of new blood vessels, regardless of mechanism. Examples ofangiogenesis inhibitors include, but are not limited to, tyrosine kinaseinhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1(VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-α (forexample Intron and Peg-Intron), interleukin-12, pentosan polysulfate,cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories(NSAIDS) like aspirin and ibuprofen as well as selectivecyclooxygenase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89,p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol.108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994); FEBS Letters,Vol. 372, p. 83 (1995); Clin. Orthop. Vol. 313, p. 76 (1995); J. Mol.Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105(1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705(1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol.274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compound of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). Examples of TAFIa inhibitors have been described in PCTPublication WO 03/013,526.

The phrase “agents that interfere with cell cycle checkpoints” refers tocompounds that inhibit protein kinases that transduce cell cyclecheckpoint signals, thereby sensitizing the cancer cell to DNA damagingagents. Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2kinases and cdk and cdc kinase inhibitors and are specificallyexemplified by 7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel)and BMS-387032.

The phrase “inhibitors of cell proliferation and survival signalingpathway” refers to agents that inhibit cell surface receptors and signaltransduction cascades downstream of those surface receptors. Such agentsinclude inhibitors of EGFR (for example gefitinib and erlotinib),antibodies to EGFR (for example C225), inhibitors of ERB-2 (for exampletrastuzumab), inhibitors of IGFR, inhibitors of cytokine receptors,inhibitors of MET, inhibitors of PI3K (for example LY294002),serine/threonine kinases (including but not limited to inhibitors of Aktsuch as described in WO 02/083064, WO 02/083139, WO 02/083140 and WO02/083138), inhibitors of Raf kinase (for example BAY-43-9006),inhibitors of MEEK (for example CI-1040 and PD-098059), inhibitors ofmTOR (for example Wyeth CCI-779), and inhibitors of C-abl kinase (forexample GLEEVEC™, Novartis Pharmaceuticals). Such agents include smallmolecule inhibitor compounds and antibody antagonists.

The phrase “apoptosis inducing agents” includes activators of TNFreceptor family members (including the TRAIL receptors).

The invention also encompasses combinations of the present compound withNSAID's which are selective COX-2 inhibitors. For purposes of thisspecification NSAID's which are selective inhibitors of COX-2 aredefined as those which possess a specificity for inhibiting COX-2 overCOX-1 of at least 100 fold as measured by the ratio of IC50 for COX-2over IC50 for COX-1 evaluated by cell or microsomal assays. Inhibitorsof COX-2 that are particularly useful in the instant method of treatmentare: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5 pyridinyl)pyridine; ora pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to, parecoxib, CELEBREX® and BEXTRA® or a pharmaceuticallyacceptable salt thereof.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the α_(v)β₅ integrin,to compounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆,α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refersto antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, STI571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1phthalazinamine, and EMD121974.

Combinations with one or more compounds that are other than anti-cancercompounds are also encompassed in the instant methods. For example,combinations of the present compound with PPAR-γ (i.e., PPAR-gamma)agonists and PPAR-δ (i.e., PPAR-delta) agonists are useful in thetreatment of certain malingnancies. PPAR-γ and PPAR-δ are the nuclearperoxisome proliferator-activated receptors γ and δ. The expression ofPPAR-γ on endothelial cells and its involvement in angiogenesis has beenreported in the literature (see J. Cardiovasc. Pharmacol. 1998;31:909-913; J. Biol. Chem. 1999;274:9116-9121; Invest. Ophthalmol Vis.Sci. 2000; 41:2309-2317). More recently, PPAR-γ agonists have been shownto inhibit the angiogenic response to VEGF in vitro; both troglitazoneand rosiglitazone maleate inhibit the development of retinalneovascularization in mice (Arch. Ophthamol. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, G1262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.

In one embodiment, useful anti-cancer (also known as anti-neoplastic)agents that can be used in combination with the present compoundinclude, but are not limited, to Uracil mustard, Chlormethine,Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin,oxaliplatin (ELOXATIN™ from Sanofi-Synthelabo Pharmaeuticals, France),Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin,Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide17α-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone,Fluoxymesterone, Dromostanolone propionate, Testolactone,Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone,Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide,Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine,Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene,Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine,Hexamethylmelamine, doxorubicin (adriamycin), cyclophosphamide(cytoxan), gemcitabine, interferons, pegylated interferons, Erbitux andmixtures thereof.

Another embodiment of the present invention is the use of the presentcompound in combination with gene therapy for the treatment of cancer.For an overview of genetic strategies to treating cancer, see Hall et al(Am J Hum Genet 61:785-789,1997) and Kufe et al (Cancer Medicine, 5thEd, pp 876-889, B C Decker, Hamilton 2000). Gene therapy can be used todeliver any tumor suppressing gene. Examples of such genes include, butare not limited to, p53, which can be delivered via recombinantvirus-mediated gene transfer (see U.S. Pat. No. 6,069,134, for example),a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of a uPA/uPARAntagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 1998;5(8):1105-13), andinterferon gamma (J Immunol 2000;164:217-222).

The present compound can also be administered in combination with one ormore inhibitor of inherent multidrug resistance (MDR), in particular MDRassociated with high levels of expression of transporter proteins. SuchMDR inhibitors include inhibitors of p-glycoprotein (P-gp), such asLY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).

The present compound can also be employed in conjunction with one ormore anti-emetic agents to treat nausea or emesis, including acute,delayed, late-phase, and anticipatory emesis, which may result from theuse of a compound of the present invention, alone or with radiationtherapy. For the prevention or treatment of emesis, the compound of thepresent invention may be used in conjunction with other anti-emeticagents, especially neurokinin-1 receptor antagonists, 5HT3 receptor,antagonists, such as ondansetron, granisetron, tropisetron, andzatisetron, GABAB receptor agonists, such as baclofen, a corticosteroidsuch as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide,Preferid, Benecorten or those as described in U.S. Pat. Nos. 2,789,118,2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and3,749,712, an antidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In one embodiment, an anti-emesis agentselected from a neurokinin-1 receptor antagonist, a 5HT3 receptorantagonist and a corticosteroid is administered as an adjuvant for thetreatment or prevention of emesis that may result upon administration ofthe present compounds.

Examples of neurokinin-1 receptor antagonists that can be used inconjunction with the present compound are described in U.S. Pat. Nos.5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270,5,494,926, 5,496,833, 5,637,699, and 5,719,147, content of which areincorporated herein by reference. In an embodiment, the neurokinin-1receptor antagonist for use in conjunction with the compounds of thepresent invention is selected from:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

The compound of the present invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

Thus, the present invention encompasses the use of the present inventivecompound (for example, for treating or preventing cellular proliferativediseases) in combination with a second compound selected from: anestrogen receptor modulator, an androgen receptor modulator, retinoidreceptor modulator, a cytotoxic/cytostatic agent, an antiproliferativeagent, a prenyl-protein transferase inhibitor, an HMG-CoA reductaseinhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δagonist, an inhibitor of inherent multidrug resistance, an anti-emeticagent, an immunologic-enhancing drug, an inhibitor of cell proliferationand survival signaling, an agent that interferes with a cell cyclecheckpoint, and an apoptosis inducing agent.

In one embodiment, the present invention empassesses the composition anduse of the present compound in combination with a second compoundselected from: a cytostatic agent, a cytotoxic agent, taxanes, atopoisomerase II inhibitor, a topoisomerase I inhibitor, a tubulininteracting agent, hormonal agent, a thymidilate synthase inhibitors,anti-metabolites, an alkylating agent, a farnesyl protein transferaseinhibitor, a signal transduction inhibitor, an EGFR kinase inhibitor, anantibody to EGFR, a C-abl kinase inhibitor, hormonal therapycombinations, and aromatase combinations.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

In one embodiment, the angiogenesis inhibitor to be used as the secondcompound is selected from a tyrosine kinase inhibitor, an inhibitor ofepidermal-derived growth factor, an inhibitor of fibroblast-derivedgrowth factor, an inhibitor of platelet derived growth factor, an MW(matrix metalloprotease) inhibitor, an integrin blocker, interferon-α,interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combretastatin A-4, squalamine,6-(O-chloroacetylcarbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, or an antibody to VEGF. In an embodiment, the estrogenreceptor modulator is tamoxifen or raloxifene.

Also included in the present invention is a method of treating cancercomprising administering a therapeutically effective amount of thecompound of Formula I in combination with radiation therapy and at leastone compound selected from: an estrogen receptor modulator, an androgenreceptor modulator, retinoid receptor modulator, a cytotoxic/cytostaticagent, an antiproliferative agent, a prenyl-protein transferaseinhibitor, an HMG-CoA reductase inhibitor, an angiogenesis inhibitor, aPPAR-γ agonist, a PPAR-δ agonist, an inhibitor of inherent multidrugresistance, an anti-emetic agent, an immunologic-enhancing drag, aninhibitor of cell proliferation and survival signaling, an agent thatinterferes with a cell cycle checkpoint, and an apoptosis inducingagent.

Yet another embodiment of the invention is a method of treating cancercomprising administering a therapeutically effective amount of thecompound of Formula I in combination with paclitaxel or trastuzumab.

The present invention also includes a pharmaceutical composition usefulfor treating or preventing cellular proliferation diseases (such ascancer, hyperplasia, cardiac hypertrophy, autoimmune diseases, fungaldisorders, arthritis, graft rejection, inflammatory bowel disease,immune disorders, inflammation, and cellular proliferation induced aftermedical procedures) that comprises a therapeutically effective amount ofthe compound of Formula I and at least one compound selected from: anestrogen receptor modulator, an androgen receptor modulator, a retinoidreceptor modulator, a cytotoxic/cytostatic agent, an antiproliferativeagent, a prenyl-protein transferase inhibitor, an HMG-CoA reductaseinhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δagonist, an inhibitor of cell proliferation and survival signaling, anagent that interferes with a cell cycle checkpoint, and an apoptosisinducing agent.

Another aspect of this invention relates to a method of selectivelyinhibiting KSP kinesin activity in a subject (such as a cell, animal orhuman) in need thereof, comprising contacting said subject with thecompound of Formula I or a pharmaceutically acceptable salt or solvatethereof.

Another aspect of this invention relates to a method of treating orpreventing a disease or condition associated with KSP in a subject(e.g., human) in need thereof comprising administering a therapeuticallyeffective amount of the compound of Formula I or a pharmaceuticallyacceptable salt or solvate thereof to said subject.

A preferred dosage is about 0.001 to 500 mg/kg of body weight/day of thecompound of Formula I or a pharmaceutically acceptable salt or solvatethereof. An especially preferred dosage is about 0.01 to 25 mg/kg ofbody weight/day of the compound of Formula I or a pharmaceuticallyacceptable salt or solvate thereof.

The phrases “effective amount” and “therapeutically effective amount”mean that amount of the compound of Formula I, and other pharmacologicalor therapeutic agents described herein, that will elicit a biological ormedical response of a tissue, a system, or a subject (e.g., animal orhuman) that is being sought by the administrator (such as a researcher,doctor or veterinarian) which includes alleviation of the symptoms ofthe condition or disease being treated and the prevention, slowing orhalting of progression of one or more cellular proliferation diseases.The formulations or compositions, combinations and treatments of thepresent invention can be administered by any suitable means whichproduce contact of these compounds with the site of action in the bodyof, for example, a mammal or human.

For administration of pharmaceutically acceptable salts of the abovecompounds, the weights indicated above refer to the weight of the acidequivalent or the base equivalent of the therapeutic compound derivedfrom the salt.

As described above, this invention includes combinations comprising anamount of the compound of Formula I or a pharmaceutically acceptablesalt or solvate thereof, and an amount of one or more additionaltherapeutic agents listed above (administered together or sequentially)wherein the amounts of the compounds/treatments result in desiredtherapeutic effect.

When administering a combination therapy to a patient in need of suchadministration, the therapeutic agents in the combination, or apharmaceutical composition or compositions comprising the therapeuticagents, may be administered in any order such as, for example,sequentially, concurrently, together, simultaneously and the like. Theamounts of the various actives in such combination therapy may bedifferent amounts (different dosage amounts) or same amounts (samedosage amounts). Thus, for illustration purposes, the compound ofFormula I and an additional therapeutic agent may be present in fixedamounts (dosage amounts) in a single dosage unit (e.g., a capsule, atablet and the like). A commercial example of such single dosage unitcontaining fixed amounts of two different active compounds is VYTORIN®(available from Merck Schering-Plough Pharmaceuticals, Kenilworth,N.J.).

If formulated as a fixed dose, such combination products employ thecompounds of this invention within the dosage range described herein andthe other pharmaceutically active agent or treatment within its dosagerange. The compound of Formula I may also be administered sequentiallywith known therapeutic agents when a combination formulation isinappropriate. The invention is not limited in the sequence ofadministration; the compound of Formula I may be administered eitherprior to or after administration of the known therapeutic agent. Suchtechniques are within the skills of persons skilled in the art as wellas attending physicians.

The pharmacological properties of the compound of this invention may beconfirmed by a number of pharmacological assays. The inhibitory activityof the present compounds towards KSP may be assayed by methods known inthe art, for example, by using the methods as described below.

While it is possible for the active ingredient to be administered alone,it is preferable to present it as a pharmaceutical composition. Thecompositions of the present invention comprise at least one activeingredient, as defined above, together with one or more acceptablecarriers, adjuvants or vehicles thereof and optionally other therapeuticagents. Each carrier, adjuvant or vehicle must be acceptable in thesense of being compatible with the other ingredients of the compositionand not injurious to the mammal in need of treatment.

Accordingly, this invention also relates to pharmaceutical compositionscomprising the compound of Formula I, or a pharmaceutically acceptablesalt or solvate thereof and at least one pharmaceutically acceptablecarrier, adjuvant or vehicle.

For preparing pharmaceutical compositions from the compound described bythis invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets and suppositories. Thepowders and tablets may be comprised of from about 5 to about 95 percentactive ingredient. Suitable solid carriers are known in the art, e.g.,magnesium carbonate, magnesium stearate, talc, sugar or lactose.Tablets, powders, cachets and capsules can be used as solid dosage formssuitable for oral administration. Examples of pharmaceuticallyacceptable carriers and methods of manufacture for various compositionsmay be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences,18^(th) Edition, (1990), Mack Publishing Co., Easton, Pa.

The term pharmaceutical composition is also intended to encompass boththe bulk composition and individual dosage units comprised of more thanone (e.g., two) pharmaceutically active agents such as, for example, acompound of the present invention and an additional agent selected fromthe lists of the additional agents described herein, along with anypharmaceutically inactive excipients. The bulk composition and eachindividual dosage unit can contain fixed amounts of the afore-said “morethan one pharmaceutically active agents”. The bulk composition ismaterial that has not yet been formed into individual dosage units. Anillustrative dosage unit is an oral dosage unit such as tablets, pillsand the like. Similarly, the herein-described method of treating asubject by administering a pharmaceutical composition of the presentinvention is also intended to encompass the administration of theafore-said bulk composition and individual dosage units.

Additionally, the compositions of the present invention may beformulated in sustained release form to provide the rate controlledrelease of any one or more of the components or active ingredients tooptimize the therapeutic effects. Suitable dosage forms for sustainedrelease include layered tablets containing layers of varyingdisintegration rates or controlled release polymeric matricesimpregnated with the active components and shaped in tablet form orcapsules containing such impregnated or encapsulated porous polymericmatrices.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection or addition of sweeteners and opacifiers fororal solutions, suspensions and emulsions. Liquid form preparations mayalso include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas,e.g. nitrogen.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally.The transdermal compositions can take the form of creams, lotions,aerosols and/or emulsions and can be included in a transdermal patch ofthe matrix or reservoir type as are conventional in the art for thispurpose.

The compounds of this invention may also be delivered subcutaneously.

Preferably the compound is administered orally.

Preferably, the pharmaceutical preparation is in a unit dosage form. Insuch form, the preparation is subdivided into suitably sized unit dosescontaining appropriate quantities of the active component, e.g., aneffective amount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 1 mg to about 100 mg, preferably fromabout 1 mg to about 50 mg, more preferably from about 1 mg to about 25mg, according to the particular application.

The actual dosage employed may be varied depending upon the requirementsof the patient and the severity of the condition being treated.Determination of the proper dosage regimen for a particular situation iswithin the skill of the art. For convenience, the total daily dosage maybe divided and administered in portions during the day as required.

The amount and frequency of administration of the compounds of theinvention and/or the pharmaceutically acceptable salts or solvatesthereof will be regulated according to the judgment of the attendingclinician considering such factors as age, condition and size of thepatient as well as severity of the symptoms being treated. A typicalrecommended daily dosage regimen for oral administration can range fromabout 1 mg/day to about 500 mg/day, preferably 1 mg/day to 200 mg/day,in two to four divided doses.

Another aspect of this invention is a kit comprising a therapeuticallyeffective amount of the compound of Formula I or a pharmaceuticallyacceptable salt or solvate thereof and at least one pharmaceuticallyacceptable carrier, adjuvant or vehicle.

Yet another aspect of this invention is a kit comprising an amount ofthe compound of Formula I or a pharmaceutically acceptable salt orsolvate thereof and an amount of at least one additional therapeuticagent listed above, wherein the amounts of the two or more ingredientsresult in desired therapeutic effect.

The KSP inhibitory activity of the compound of Formula I was measured bythe following assays.

KSP Assays:

Endpoint Assay:

Serial dilutions of the compounds were prepared in a low binding,96-well microtiter plate (Costar # 3600) using 40% DMSO (Fisher BP231).The diluted compounds were added to a 384-well microtiter plate (Fisher12-565-506). The following was then added to each well of the 384microtiter plate: 55 μg/mL purified microtubules (Cytoskeleton TL238),2.5-10 nM KSP motor domain [made according to Hopkins et al,Biochemistry, (2000) 39, 2805-2814)], 20 mM ACES pH 7.0 (Sigma A-7949),1 mM EGTA (Sigma E-3889), 1 mM MgCl₂ (Sigma M-2670), 25 mM KCl (SigmaP-9333), 10 μM paclitaxel (Cytoskeleton TXD01), and 1 mM DTT (SigmaD5545) [final concentration]. Following a 10 minute incubation, ATP(Sigma A-3377) (final concentration of ATP: 100 μM) was added to startthe reaction. The final reaction volume was 25 μL. Final test compoundconcentration ranged from 50 μM to 5 nM. The reaction was incubated for1 hour at room temperature. The reaction was stopped by the addition of50 μL Biomol green reagent (Biomol AK111) per well, and was allowed toincubate for 20 minutes at room temperature. The 384-well microtiterplate was then transferred to an absorbance reader (Molecular DevicesSpectraMax plus) and a single measurement was taken at 620 nm.

Kinetic Assay:

Compound dilutions were prepared as described previously. 25A25 bufferconsisted of the following: 25 mM ACES pH 6.9, 2 mM MgOAc (SigmaM-9147), 2 mM EGTA, 0.1 mM EDTA (Gibco 144475-038), 25 mM KCl, 1 mM2-mercaptoethanol (Biorad 161-0710), 10 μM paclitaxel, and 0.5 mM DTT.Solution 1 consisted of the following: 3.75 mM [final concentration]phosphoenol pyruvic acid (PEP, 2.5×) (Sigma P-7127), 0.75 mM MgATP(2.5×) (Sigma A-9187) in 1×25A25 buffer. Solution 2 consisted of thefollowing: 100-500 nM KSP motor domain (2×), 6 U/mL pyruvatekinase/lactate dehydrogenase (2×) (Sigma P-0294), 110 μg/mL purifiedmicrotubules (2×), 1.6 μM β-nicotinamide adenine di-nucleotide, reducedform (NADH, 2×) (Sigma N-8129) in 1×25A25 buffer. Compound dilutions [8]were added to a 96-well microtiter plate (Costar 9018), and 40 μL ofsolution 1 was added to each well. The reaction was started by adding 50μL of solution 2 to each well. The respective final assay concentrationswere: 1.5 mM PEP, 0.3 mM MgATP, 50-250 nM KSP motor domain, 3 U/mLpyruvate kinase/lactate dehydrogenase, 55 μg/mL purified microtubules,0.8 μM NADH [final concentrate]. The microtiter plate was thentransferred to an absorbance reader and multiple readings were taken foreach well in a kinetic mode at 340 nm [25 measurements for each wellapprocimately every 12 seconds, spread approximately over about 5minutes time span]. For each reaction, a rate of change was determined.

Calculations:

For both endpoint and kinetic assays, the percent activity for eachconcentration is calculated using the following equation:Y=((X−background)/(positive control−background))*100

-   -   Y is the % activity and X is the measured reading (OD620 or        rate)

For an IC₅₀ determination, the % activity was fit by the followingequation using a nonlinear curve-fitting program for sigmoidaldose-responses (variable slopes) (GraphPad Prizm).Y=Bottom+(Top−Bottom)/(1+10^((LogEC50−X)*HillSlope))

-   -   X is the logarithm of concentration. Y is the response.    -   Y starts at Bottom and goes to Top with a sigmoid shape.

REFERENCES

KSP/Kinesin as Target

-   1) Blangy, A et al. (1995) Cell 83, 1159-1169 (cloning of human KSP,    function in mitosis).-   2) Sawin, K. and Mitchison, T. J. (1995) Proc. Natl. Acad. Sci. 92,    4289-4293 (Xenopus Egd5, conserved motor domain, function).-   3) Huang, T.-G. and Hackney, D. D. (1994) J. Biol. Chem. 269,    16493-16501 (Drosphila kinesin minimal motor domain definition,    expression and purification from E. coli).-   4) Kaiser A. et al. (1999) J. Biol. Chem. 274, 18925-18931    (overexpression of KSP motor domain, function in mitosis, inhibition    of growth by targeting KSP).-   5) Kapoor T. M and Mitchison, T. J. (1999) Proc. Natl. Acad. Sci.    96, 9106-9111 (use of KSP motor domain, inhibitors thereof.-   6) Mayer, T. U. (1999) Science 286, 971-974 (KSP inhibitors as    anticancer drugs).    KSP Assays (Endpoint and Kinetics)-   7) Wohlke, G. et al. (1997) Cell 90, 207-216 (expression and    purification of kinesin motor domain, kinetics assay, endpoint    assay).-   8) Geladeopoulos, T. P. et al. (1991) Anal. Biochem. 192, 112-116    (basis for endpoint assay).-   9) Sakowicz, R. et al. (1998) Science 280, 292-295 (kinetics assay).-   10) Hopkins, S. C. et al. (2000) Biochemistry 39, 2805-2814    (endpoint and kinetics assay).-   11) Maliga, Z. et al. (2002) Chem. & Biol. 9, 989-996 (kinetics    assay).

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

1. A pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, adjuvant or vehicle:


2. The composition of claim 1, wherein the compound is the (+)-enantiomer of

or a pharmaceutically acceptable salt thereof.
 3. The composition of claim 1, wherein the compound is the (−)-enantiomer of

or a pharmaceutically acceptable salt thereof.
 4. The pharmaceutical composition of claim 1, further comprising one or more compounds selected from the group consisting of an anti-cancer agent, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of inherent multidrug resistance, an anti-emetic agent, and an immunologic-enhancing drug.
 5. The pharmaceutical composition of claim 4, wherein the anti-cancer agent is selected from the group consisting of an estrogen receptor modulator, an androgen receptor modulator, retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an angiogenesis inhibitor, an inhibitor of cell proliferation and survival signaling, an agent that interferes with a cell cycle checkpoint, and an apoptosis inducing agent.
 6. The pharmaceutical composition of claim 4, further comprising one or more anti-cancer agents selected from the group consisting of cytostatic agents, cytotoxic agents, taxanes, topoisomerase II inhibitors, topoisomerase I inhibitors, tubulin interacting agents, hormonal agents, thymidilate synthase inhibitors, anti-metabolites, alkylating agents, farnesyl protein transferase inhibitors, signal transduction inhibitors, EGER kinase inhibitors, antibodies to EGER, C-abl kinase inhibitors, hormonal therapy combinations, and aromatase combinations.
 7. The pharmaceutical composition of claim 4, further comprising one or more agents selected from the group consisting of Uracil mustard, Chlormethine, Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin, oxaliplatin, Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide 17α-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methyipred nisolone, Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene, Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine, Hexamethyl melamine, doxorubicin, cyclophosphamide, gemcitabine, interferons, pegylated interferons, Erbitux and mixtures thereof. 